The thrombin-thrombomodulin (TM) interaction is critical for the cessation of blood clotting and maintenance of hemostasis. Structures of both proteins are now available, but much still needs to be learned about the dynamics of this important interaction. A comprehensive structure-function study is proposed in which each protein will be studied individually (Aims 1 and 2), the mechanism of their association will be analyzed (Aim 3) and finally, the activity of the thrombin-TM complex towards protein C will be probed (Aim 4). In Aim 1, we shall study how the two essential EGF-like domains (EGF4 and EGF5) work together to cause anticoagulant cofactor activity. Our hypothesis is that the two domains of TM, a non-globular protein, communicate via structural and dynamic effects and are tightly coupled. The solution structure and backbone dynamics will be correlated with specific kinetic parameters (KM for thrombin, KM for protein C, kcat for protein C activation). The role of the 6th domain, which aids in thrombin binding, shall also be explored. In Aim 2, conformational changes in thrombin that occur when TM binds will be studied to see whether they are important for catalysis of protein C activation. Our recently developed method for probing changes in backbone amide exchange has revealed beta-sheets within thrombin that tighten upon TM binding, and these "transmission lines" connect the TM binding site to the active site. Various substrate analogs and TM fragments devoid of cofactor activity will be used to discover which transmission lines correlate with anticoagulant activity. In Aim 3, the thermodynamic and kinetic driving forces for thrombin-TM association will be probed using surface plasmon resonance on mutants of both proteins. In Aim 4, protein C activation by the thrombin-TM complex will be probed. Our hypothesis is that TM does more than provide a binding scaffold for protein C. A panel of mutants will be analyzed to determine which residues cause specific defects (KM for thrombin, KM for protein C, kcat for thrombin activation of protein C). Those that show kcat defects will be used to further delineate which transmission lines in thrombin are important for anticoagulant cofactor activity.